The present invention relates to methods for the production of high titers of serum-free lytic viruses in a hollow fiber cartridge capillary system. The invention further relates to methods of infecting target cells at high multiplicity and for producing high concentrations of transduced target cells.
Gene therapy is defined as the use of genetic material for therapeutic purposes. Patients diagnosed with inheritable genetic diseases can be, in principle, treated by the introduction of a therapeutic gene in replacement of an aberrantly functioning gene. For example, aberrant functioning of the adenosine deaminase (ADA) gene, the gene which encodes the low density protein in familial hypercholesterolemia and the chloride transporter gene in Cystic Fibrosis can be corrected by the introduction of therapeutic genes.
There also are examples where overexpression of an oncogene, or the lack of expression of a tumor suppressor, could be the molecular basis of cancers. Under these conditions, both corrective as well as cytoreductive strategies have been used to introduce genetic materials, either to repair the damage that caused aberrant growth of the cancer cells, or the use of a cytoreductive strategy to introduce various forms of toxic genes that can control the growth of cancer cells. For these reasons, gene therapy has become increasingly popular for the treatment of inheritable genetic diseases, as well as cancer and cardiovascular diseases. As of May 12, 1998, more than 212 gene therapy protocols have been approved by the Recombinant DNA and Advisory Committee (RAC) of the National Institute of Health (NIH) since the first human ADA deficient patients receives such therapy in 1990. In the approved protocols by the RAC of the NIH, 147 out of 212 approved protocols are for the treatment of cancer using a gene therapeutic approach.
The current invention describes a novel method whereby high yields (6xc3x971012 virus particles) of lytic-viral vectors can be produced consistently and reproducibly. This method is an improvement over the current method for the production of clinical therapeutic grade viral vectors because the entire production process is performed in a self-contained system and in a serum-free condition using a hollow fiber bioreactor system. This new technique has the further advantage of allowing scale-up to a level that is not economically or physically feasible using the current technique.
Prior to the instant invention, similar techniques were performed only on retroviral vectors, and not on lytic-viral vectors, as disclosed in the present application. See, U.S. Pat. No. 5,498,537, entitled xe2x80x9cSerum-free production of packaged viral vectorxe2x80x9d.
The present invention significantly differs from what was previously known in the art. For example, the present invention relates to the production of lytic-viral vectors whereas previously, only methods of utilizing budding retroviral vectors were known. A further difference between what was previously known in the art and the instant invention is that previously, PA 117 derived packaging cells were principally used as hosts for the production of retroviral vectors, whereas the present application uses human fetal kidney 293 cells for the production of lytic-viral vectors. Although artificial capillary hollow fiber cartridge systems were previously known in the art, it was not until the instant invention that completely serum-free medium was used to store cells. Prior to the instant invention, packaged cell lines typically were cultured initially in the presence of bovine serum-containing culture medium, and then subsequently either continued in serum-containing media or the serum-containing medium was replaced with a serum-free medium. As noted above, the instant application describes a 293 cell culture that has been adapted to grow in serum-free medium, and can be stored under serum-free conditions with 10% DMSO. This condition is favorable in that the culture cells can be retrieved quickly from liquid nitrogen, and subjected to culture immediately, without needing additional adaptation steps. Further, using this technique, cell cultures have good viability and grow under completely serum-free conditions. This allows the viral infection to occur quickly and reproducibly and avoids the potential contamination from serum preparations. Previously, retroviral-producing cell lines which were capable of producing retroviruses before seeding the capillary system were used. The current methodology, to the contrary, describes the use of 293 cells grown under serum-free conditions at early onset that are subjected to infection by the master copy of the lytic virus after growth in the capillary system has been established to an appropriate level of lactate production.
Further, prior methods involved harvesting retrovirus from the medium of the hollow fiber cartridge system where the budding retroviruses reside, whereas the current methods involve the harvesting of adenoviruses from both the cells as well as the medium within the extracapillary space (ECS).
The large scale production of recombinant adenoviruses is based mostly on a book chapter written by Graham, F. L. and L. Prevec (1991). Manipulation of adenovirus vectors. Clifton, N.J., The Humana Press, inc. and updated article by Frank, F. L. and Prevec, L. Methods for construction of adenovirus vectors, Molecular Biotechniques, 3:207-220, 1995.
The present invention generally relates to lytic viruses. More particularly, the invention is directed to a novel method for the production of lytic viruses using a capillary system. After infecting host cells with a lytic virus, the infected host cells are cultured and the lytic virus is harvested from the culture medium.
In a preferred embodiment, the lytic viruses of the invention are either replication defective or replication competent. In other embodiments, the lytic viruses of the invention include, but are not limited to, adenoviruses, parvoviruses, adeno-associated viruses, herpes simplex viruses, polio viruses or papillomaviruses. In further embodiments of the invention, the host cells include, but are not limited to, 293 cells, CCL81.1 cells, Vero cells, HEL cells or BHK cells.
In a further preferred embodiment, the capillary system is a hollow fiber cartridge system. In yet other embodiments of the invention, the virus is a viral vector, and, further, the viral vector encodes a therapeutically effective product. Another preferred embodiment of the invention involves host cells which have been adapted to grow and be stored under serum-free conditions. Specifically, an example of a serum-free condition is storage of host cells in 10% DMSO.
In another embodiment, viral infection is carried out at a cellular lactate production rate of 500 mg/day. Furthermore, viruses are harvested at a cellular lactate production rate of between 1000 and 1500 mg/day. A further embodiment of the invention relates to host cells which are substantially isolated from the cellular material. In still a further embodiment of the invention, the viruses are harvested from the extracapillary space of the capillary system.